Project Summary/Abstract Clostridium difficile is a leading cause of diarrhea in hospitalized patients and infection is acquired by ingestion of spores that germinate into toxin-producing vegetative forms that destroy colonic epithelial integrity. Intestinal inflammation induced by C. difficile requires spore germination within the GI tract, bacterial proliferation and toxin production leading to intestinal epithelial damage. C. difficile growth in the colon occurs when the composition of the commensal bacterial flora is damaged by antibiotic treatment. Specific members of the commensal flora inhibit C. difficile growth, in part by converting primary to secondary bile salts. Once C. difficile infection has damaged the colonic epithelial layer, the host?s immune system is activated and, while essential for host survival, also contributes to intestinal pathology. Inflammatory monocytes, neutrophils, innate lymphocytes (ILCs) and a range of inflammatory cytokines have been implicated in defense against C. difficile infection (CDI) and in inflammatory pathology. The goal of our studies is to identify and characterize microbiota and immune-mediated protective mechanisms and to test our discoveries on microbiologically and clinically diverse C. difficile strains. Our specific aims are: 1.) To assemble minimal-complexity commensal bacterial consortia derived from human fecal samples that provide high-level resistance against CDI. 2.) To characterize microbiota-mediated mechanisms that promote ILC1-mediated resistance against C. difficile and to test the hypothesis that the residual microbiota determines the balance of ILC1 versus ILC3 differentiation. 3.) To determine whether microbiota or ILC1 mediated defenses against CDI differ for distinct C. difficile strains. The proposed studies will provide novel insights into microbiota-mediated defenses against CDI and will also identify immune mechanisms that ameliorate adverse inflammatory responses during early CDI. These insights will facilitate the development of therapies to prevent and treat C. difficile infections.